Peptide array quality control

ABSTRACT

The present application provides arrays for use in immunosignaturing and quality control of such arrays. Also disclosed are peptide arrays and uses thereof for diagnostics, therapeutics and research.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/799,494, filed on Mar. 15, 2013, which is incorporated by referenceherein in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.HDTRA1-11-1-0010 and Contract No. HDTRA1-12-C-0058 awarded by theDefense Threat Reduction Agency. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Screening mechanisms may be used for assessing samples for changes inpolynucleotide and/or polypeptide concentration and/or expression. Whilethere are some peptide arrays available commercially, such arrays havelow density and relatively low fidelity.

SUMMARY OF THE INVENTION

Disclosed herein are methods, components and compositions fordetermining the quality of a biopolymer array of interest in the contextof performing diagnostic assays. The methods, components andcompositions disclosed herein can be used to assess or determine if abiopolymer array has sufficient fidelity, density and/or othercharacteristics necessary for performing diagnostic assays on thebiopolymer arrays. The disclosed embodiments can be used as a primaryquality control method, or can be used in conjunction with other qualitycontrol methods for assessing the quality of a biopolymer array ofinterest.

Accordingly, disclosed herein are methods for determining the quality ofa biopolymer array of interest. The biopolymer array may comprise aplurality of molecules coupled to the surface of the array, wherein themolecules are synthesized in situ. In some embodiments, the methods canbe used to determine the quality of a biopolymer array prior toinitiating a diagnostic procedure on the array. In other embodiments,the methods can be used to determine the quality of a biopolymer arrayconcurrently with a diagnostic procedure, for example, for assessing theimmunosignature of a subject or patient. In some embodiments, thesubject is a mammal, avian or reptile. In other embodiments, the subjectis human. In still other embodiments, the subject is a livestock animal,e.g., bovine, porcine or ovine species, dog, cat, bird, or otherdomestic or wild animal.

In one aspect, the methods, components and compositions disclosed hereinprovide for the assessment of the quality of a biopolymer array ofinterest by providing an array of compounds, incubating the biopolymerarray with a biological sample, detecting target binding of thebiological components on the array, determining the average targetbinding values of the biological components to the compounds on thearray, and assessing the quality of the array from the average targetbinding values obtained.

In another aspect, disclosed herein are methods for determining thequality of a biopolymer array of interest, the methods comprising:

-   -   a. providing an array of compounds, wherein the compounds are        synthesized in situ in a stepwise manner on the surface of the        biopolymer array;    -   b. incubating a biological sample on the biopolymer array;    -   c. detecting target binding on individual compounds on the        biopolymer array;    -   d. determining the average target binding values of individual        compounds affected at each synthesis step; and    -   f. comparing the average target binding values obtained at each        synthesis step to a universal average intensity value for the        biopolymer array;    -   wherein a difference of less than a threshold level between the        average target binding values at each synthesis step and the        universal average intensity value indicates a failure of the        synthesis step in synthesizing the molecules on the array.

In one embodiment, the threshold level is a percent difference betweenthe average target binding values at each synthesis step and theuniversal average intensity value obtained. In another embodiment, thethreshold level indicating failure of the synthesis step is less thanabout 2%, less than about 5%, less than about 10%, less than about 20%,less than about 25% or less than about 30%. In another embodiment, thethreshold level indicating failure of the synthesis step is less thanabout 20%. In still another embodiment, the threshold level indicatingfailure of the synthesis step is less than about 10%. In yet anotherembodiment, the threshold level indicating failure of the synthesis stepis less than about 5%.

In some embodiments, the threshold level is a difference in standarderror between the average target binding values at each synthesis stepand the universal average intensity value obtained. In one embodiment,the threshold level indicating failure of the synthesis step is lessthan 4× the standard error (SE), less than 3.5× SE, less than 3× SE,less than 2.5× SE or less than 2× SE. In another embodiment, thethreshold level indicating failure of the synthesis step is less than 3×SE. In still other embodiments, the threshold level indicating failureof the synthesis step is less than 2.5× SE. In yet other embodiments,the threshold level indicating failure of the synthesis step is lessthan 2× SE.

In some embodiments, the biopolymer array is a peptide array or apolynucleotide array. In other embodiments, the biopolymer array is apeptide array. In still other embodiments, the peptide array is a randompeptide array.

In some embodiments, the compound is a nucleic acid, a peptide, apolymer, a lipid, a polysaccharide or an analog thereof. In still otherembodiments, the compound is a peptide. In yet other embodiments, thepeptides are synthesized in situ on the array surface. In someembodiments, the peptide length is between 10-25 amino acid residues. Instill other embodiments, the peptide length is between 15-20 amino acidresidues. In yet other embodiments, the peptide spacing within a featureon the array is less than 6 nm, less than 4 nm or less than 2 nm. In yetother embodiments, the biological sample is a blood sample, a serumsample, a plasma sample, a urine sample, a sputum sample, a mucosalsample, a peritoneal fluid sample, a tissue sample, an exudates, aneffusion or a biopsy.

Also disclosed herein are methods for determining the quality of abiopolymer array of interest, the methods comprising:

-   -   a. providing an array of compounds, wherein the compounds are        synthesized in situ in a stepwise manner on the surface of the        biopolymer array;    -   b. incubating a biological sample on the biopolymer array;    -   c. detecting target binding on individual compounds on the        biopolymer array;    -   d. determining the average target binding values of individual        compounds affected at each synthesis step; and    -   e. comparing the average target binding values obtained at each        synthesis step to a universal average intensity value for the        biopolymer array;    -   wherein a difference of greater than a threshold level between        the average target binding values at each synthesis step and the        universal average intensity value indicates fidelity of the        synthesis step and acceptable synthesis of the compounds on the        array.

In some embodiments, the threshold level is a percent difference betweenthe average target binding values at each synthesis step and theuniversal average intensity value obtained. In some embodiments, thethreshold level indicating fidelity of the synthesis step is at leastover 2%, at least over 3%, at least over 4%, at least over 5%, at leastover 6%, at least over 7%, at least over 8%, at least over 9%, at leastover 10%, at least over 15%, at least over 20%, at least over 25%, or atleast over 30%. In yet other embodiments, the threshold level indicatingfidelity of the synthesis step is at least over 25%. In still otherembodiments, the threshold level indicating fidelity of the synthesisstep is at least over 10%. In one embodiment, the threshold levelindicating fidelity of the synthesis step is at least over 5%.

In some embodiments, the threshold level is a difference in standarderror between the average target binding values at each synthesis stepand the universal average intensity value obtained. In one embodiment,the threshold level indicating fidelity of the synthesis step is atleast over 4× the standard error (SE), at least over 3.5× SE, at leastover 3× SE, at least over 2.5× SE or at least over 2× SE. In anotherembodiment, the threshold level indicating fidelity of the synthesisstep is at least over 3× SE. In other embodiments, the threshold levelindicating fidelity of the synthesis step is at least over 2.5× SE. Inyet other embodiments, the threshold level indicating fidelity of thesynthesis step is at least over 2× SE.

A method for determining the quality of a biopolymer array of interest,comprising:

-   -   a. providing an array of compounds, wherein the compounds are        synthesized in situ in a stepwise manner by sequential addition        of different components at a specific position in b. the        compound on the surface of the biopolymer array;    -   c. incubating a biological sample on the biopolymer array;    -   d. detecting target binding on individual compounds on the        biopolymer array;    -   e. determining the average target binding values of each        component at a specific position in individual compounds        synthesized on the array; and    -   f. comparing the average target binding values obtained for each        component at each position in individual compounds synthesized        to a universal average intensity value for the biopolymer array;    -   wherein a difference of less than a threshold level between the        average target binding values for each component at each        position in individual compounds synthesized and the universal        average intensity value indicates a failure of a synthesis step        for the compounds on the array.

In one embodiment, the threshold level is a percent difference betweenthe average target binding values at each position in individualcompounds synthesized and the universal average intensity valueobtained. In some embodiments, the threshold level indicating failure ofthe synthesis step is less than about 2%, less than about 5%, less thanabout 10%, less than about 20%, less than about 25% or less than about30%. In yet other embodiments, the threshold level indicating failure ofthe synthesis step is less than about 20%. In still other embodiments,the threshold level indicating failure of the synthesis step is lessthan about 10%. In yet other embodiments, the threshold level indicatingfailure of the synthesis step is less than about 5%.

In some embodiments, the threshold level is a difference in standarderror between the average target binding values at each position inindividual compounds synthesized and the universal average intensityvalue obtained. In other embodiments, the threshold level indicatingfailure of the synthesis step is less than 4× the standard error (SE),less than 3.5× SE, less than 3× SE, less than 2.5× SE or less than 2×SE. In still other embodiments, the threshold level indicating failureof the synthesis step is less than 3× SE. In still other embodiments,the threshold level indicating failure of the synthesis step is lessthan 2.5× SE. In still other embodiments, the threshold level indicatingfailure of the synthesis step is less than 2× SE.

In some embodiments, the biopolymer array is a peptide array or apolynucleotide array. In yet other embodiments, the biopolymer array isa peptide array. In some embodiments, the peptide array is a randompeptide array. In yet other embodiments, the compound is a nucleic acid,a peptide, a polymer, a lipid, a polysaccharide or an analog thereof. Instill other embodiments, the compound is a peptide.

In yet other embodiments, the peptides are synthesized in situ on thearray surface. In still other embodiments, the peptide length is between10-25 amino acid residues. In yet other embodiments, the peptide lengthis between 15-20 amino acid residues. In one embodiment, the peptidespacing within a feature on the array is less than 6 nm, less than 4 nmor less than 2 nm. In another embodiment, the biological sample is ablood sample, a serum sample, a plasma sample, a urine sample, a sputumsample, a mucosal sample, a peritoneal fluid sample, a tissue sample, anexudates, an effusion or a biopsy.

Also disclosed herein are methods for determining the quality of abiopolymer array of interest, comprising:

-   -   a. providing an array of compounds, wherein the compounds are        synthesized in situ in a b. stepwise manner by sequential        addition of different components at a specific position in the        compound on the surface of the biopolymer array;    -   c. incubating a biological sample on the biopolymer array;    -   d. detecting target binding on individual compounds on the        biopolymer array;    -   e. determining the average target binding values of each        component at a specific position in individual compounds        synthesized on the array; and    -   f. comparing the average target binding values obtained for each        component at each position in individual compounds synthesized        to a universal average intensity value for the biopolymer array;    -   wherein a difference of greater than a threshold level between        the average target binding values for each component at each        position in individual compounds synthesized and the universal        average intensity value indicates a fidelity of a synthesis step        for the compounds on the array.

In one embodiment, the threshold level is a percent difference betweenthe average target binding values at each position in individualcompounds synthesized and the universal average intensity valueobtained. In another embodiment, the threshold level indicating fidelityof the synthesis step is at least over 2%, at least over 3%, at leastover 4%, at least over 5%, at least over 6%, at least over 7%, at leastover 8%, at least over 9%, at least over 10%, at least over 15%, atleast over 20%, at least over 25%, or at least over 30%. In yet anotherembodiment, the threshold level indicating fidelity of the synthesisstep is at least over 25%. In still another embodiment, the thresholdlevel indicating fidelity of the synthesis step is at least over 10%. Inyet another embodiment, the threshold level indicating fidelity of thesynthesis step is at least over 5%.

In one embodiment, the threshold level is a difference in standard errorbetween the average target binding values at each synthesis step and theuniversal average intensity value obtained. In one embodiment, thethreshold level indicating fidelity of the synthesis step is at leastover 4× the standard error (SE), at least over 3.5× SE, at least over 3×SE, at least over 2.5× SE or at least over 2× SE. In another embodiment,the threshold level indicating fidelity of the synthesis step is atleast over 3× SE. In still another embodiment, the threshold levelindicating fidelity of the synthesis step is at least over 2.5× SE. Inyet another embodiment, the threshold level indicating fidelity of thesynthesis step is at least over 2× SE.

Also disclosed herein are methods for determining the quality of abiopolymer array of interest, comprising:

-   -   a. obtaining a first binding pattern of the array of interest;    -   b. obtaining a second binding pattern of a reference array;    -   c. comparing the first binding pattern with the second binding        pattern and calculating a correlation value; and    -   d. determining the quality of the array,

wherein a correlation value of less than a threshold level indicates afidelity of the biopolymer array.

In one embodiment, the correlation value is a correlation coefficient.In another embodiment, the correlation coefficient is obtained byPearson Correlation or Spearman rank correlation, or Kolmogorov-Smirnovtest. In another embodiment, the threshold level is defined as apercentage of the correlation coefficient between assays.

In one embodiment, the threshold level is at least under 3%, at leastunder 4%, at least under 5%, at least under 6%, at least under 7%, atleast under 8%, at least under 9%, at least under 10%, at least under15%, at least under 20%, at least under 25%, at least under 30%, atleast under 35%, at least under 40%, at least under 45%, or at leastunder 50%. In yet another embodiment, the threshold level is at leastunder 3%. In still another embodiment, the threshold level is at leastunder 5%. In still another embodiment, the threshold level is at leastunder 10%. In yet another embodiment, the threshold level is at leastunder 15%.

In one embodiment, the threshold level is defined as a standarddeviation derived from calculation of a plurality of correlationcoefficients between an assay and a standard assay. In anotherembodiment, the threshold level is at least under 2× SD, at least under2.5× SD, at least under 3× SD, at least under 3.5× SD or at least under4× SD. In still another embodiment, the threshold level is at leastunder 2× SD. In yet another embodiment, the threshold level is at leastunder 3× SD. In still another embodiment, the threshold level is atleast under 4× SD.

In one embodiment, the first binding pattern is obtained from a subsetof biopolymers on the array of interest, and the second binding patternis obtained from a corresponding subset of biopolymers on the referencearray. In another embodiment, the first binding pattern is obtained frombinding with a first sample, the second binding pattern is obtained frombinding with a second sample, wherein the first sample and the secondsample are from the same species. In yet another embodiment, the firstsample and second sample are not from the same subject. In still anotherembodiment, the species is human being. In yet another embodiment, thefirst sample and second sample are a blood sample.

Also included herein are methods for determining the quality of a batchof biopolymer arrays, comprising:

-   -   a. obtaining a binding pattern of at least two arrays of a        batch;    -   b. obtaining a correlation value of the binding pattern between        the at least two arrays; and    -   c. comparing the correlation value with a correlation range.        wherein a correlation value of less than a threshold level        indicates a fidelity of the biopolymer array.

In one embodiment, the correlation values of the binding patterns areobtained between all possible pairs of arrays of the batch. In anotherembodiment, the correlation value is a correlation coefficient. In stillanother embodiment, the correlation coefficient is obtained by PearsonCorrelation or Spearman rank correlation, or Kolmogorov-Smirnov test.

In one embodiment, the threshold level is defined as a percentage of thecorrelation coefficient between assays. In yet another embodiment, thethreshold level is at least under 3%, at least under 4%, at least under5%, at least under 6%, at least under 7%, at least under 8%, at leastunder 9%, at least under 10%, at least under 15%, at least under 20%, atleast under 25%, at least under 30%, at least under 35%, at least under40%, at least under 45%, or at least under 50%. In still anotherembodiment, the threshold level is at least under 3%. In still anotherembodiment, the threshold level is at least under 5%. In yet anotherembodiment, the threshold level is at least under 10%. In yet anotherembodiment, the threshold level is at least under 15%.

In one embodiment, the threshold level is defined as a standarddeviation derived from calculation of a plurality of correlationcoefficients between an assay and a standard assay. In yet anotherembodiment, the threshold level is at least under 2× SD, at least under2.5× SD, at least under 3× SD, at least under 3.5× SD or at least under4× SD. In yet another embodiment, the threshold level is at least under2× SD. In yet another embodiment, the threshold level is at least under3× SD. In still another embodiment, the threshold level is at leastunder 4× SD.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.If more than one version of a sequence is associated with a depositnumber at different times, the version associated with the depositnumber at the effective time of filing the application is meant.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates average intensity vs. step number (mask number).

FIG. 2 flowchart illustrating actors and processes used in imageacquisition, alignment, translation, and analysis.

FIG. 3 is a block diagram illustrating a first-example architecture of acomputer system that can be used in connection with example embodimentsof the present invention.

FIG. 4 is a diagram illustrating a computer network that can be used inconnection with example embodiments of the present invention.

FIG. 5 is a block diagram illustrating a second example architecture ofa computer system that can be used in connection with exampleembodiments of the present invention.

FIG. 6 is a diagram of a 1-box computer farm capable of integrating withthe present invention, ideally supporting all computer needs in a singletuned system.

FIG. 7 is a statistical correlation utilized in a quality controlanalysis.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Specific binding refers to the binding of a compound to a target (e.g.,a component of a sample) that is detectably higher in magnitude anddistinguishable from non-specific binding occurring to at least oneunrelated target. Specific binding can be the result of multiple,structurally specific interactions between particular chemical groups inthe ligand and its binding partner or a particular spatial fit (e.g.,lock and key type) whereas nonspecific binding is usually the result ofinteractions in which there is not particular spatial arrangement of theligand relative to its binding partner (e.g., general hydrophobic orcharge-charge interactions that do not result in a specific structure ofthe binding pair). Specific binding does not however imply that acompound binds one and only one target. Thus, a compound can and oftendoes show specific binding of different strengths to several differenttargets and only nonspecific binding to other targets. Preferably,different degrees of specific binding can be distinguished from oneanother as can specific binding from nonspecific binding. Specificbinding often involves an apparent association constant of 10³ orhigher, where the concentrations of components in the unit-lessassociation constant are defined relative to their standard states (thisis true for all association constant values listed in thisspecification).

An association constant is an equilibrium constant for the bindingreaction between a ligand (L) and its binding partner (B): L+B←→LB,

$K_{assoc} = \frac{\left( \frac{LB}{LB_{0}} \right)}{\left( {\frac{L}{L_{0}} \times \frac{B}{B_{0}}} \right)}$where L, B and LB in this equation are the concentrations of the ligand,binding partner and the ligand/binding partner complex, respectively,and L₀, B₀ and LB₀ are the standard state concentrations of ligand,binding partner and the ligand/binding partner complex, respectively.The dissociation constant for the same binding interaction would begiven by 1/K_(assoc). The term “apparent association constant” refers tothe value calculated for K_(assoc) as defined above under conditionswhere one or more binding partner molecules are confined spatially, suchas on a surface, such that multiple binding partners are in close enoughproximity to act in concert or the confined environment, such as asurface alters the nature of the interaction between the ligand and thebinding partner. The term “apparent dissociation constant” refers to theinverse of the apparent association constant. In the text contained inthis document, the use of the terms “association constant” or“dissociation constant” may include both true and apparent associationand dissociation constants.

Specific binding can additionally or alternatively be defined as abinding strength (e.g., fluorescence intensity) more than three standarddeviations greater than background represented by the mean bindingstrength of empty control areas in an array (i.e., having no compound,where any binding is nonspecific binding to the support). The range ofaffinities or avidities of compounds showing specific binding to amonoclonal or other sample can vary by from about 1 to about 4 and oftenfrom about 2.5 to about 3.5 orders of magnitude. An apparent associationconstant includes avidity effects if present (in other words, if atarget shows enhanced affinity to multiple molecules of the samecompound, the apparent association constant is a value reflecting theaggregate binding of the multiple molecules of the same compound to thetarget). When contacted with a random selection of monoclonalantibodies, a subset of compounds (e.g., from about 1 to about 20, orfrom about 5 to about 15%) have association constants in the range offrom about 10³ to about 10⁶, from about 2×10³ to about 10⁶ or from about10⁴ to about 10⁶ to at least one and sometimes several (e.g., at leastabout 2, about 5 or about 10) different targets. A subset of allpeptides or other compounds (e.g., at least about 1%, at least about 5%or about 10%; from about 1 to about 75%, from about 5 to about 60%, fromabout 1 to about 20% or from about 5 to about 15%) usually shows actualassociation constants of from about 10³ to about 10⁶ to at least one andusually several targets (e.g., at least about 2, about 5 or about 10).The same ranges of association constant apply to composite targetsbinding to the same compound in a complex sample. Of course differentcompounds in an array have different degrees of binding strength tocomponents of a sample and some compounds can bind with higher or lowerapparent association constants than these ranges.

Avidity is defined as enhanced binding of a component in solution to asurface that includes multiple copies of a compound, such as a peptide,that the solution component has affinity for. In other words, given acompound on a surface that individually has some affinity for acomponent of a solution, avidity reflects the enhanced apparent affinitythat arises when multiple copies of the compound are present on thesurface in close proximity. Avidity is distinct from cooperative bindingin that the interaction does not involve simultaneous binding of aparticular molecule from the solution to multiple molecules of thecompound on the surface. Avidity interactions and/or cooperative bindingcan occur during the association of components of a solution, such asantibodies in blood, with compounds on a surface.

Patients include humans, veterinary animals, such as cats, dogs, horses,farm animals, such as chickens, pigs, sheep, cattle and laboratoryanimals, such as rodents, e.g., mice and rats.

A binding profile of an array is a measure of the amount of component(s)of a sample bound to the different compounds of an array to a particularsample. The amount of component(s) bound reflects the amount of thecomponents in the sample as well as the binding strength of componentsto the compounds. A binding profile can be represented for example as amatrix of binding strengths corresponding to the different compounds inan array. A binding profile typically includes binding strengths of aplurality of compounds (e.g., at least 2, 10, 50, 100 or 1000 havingdissociation constants in a range of from about 10⁻³ to about 10⁻⁶ to asample).

Binding strength can be measured by association constant, dissociationconstant, dissociation rate, or association rate, or a composite measureof affinity which may include one or more of these measures. Thestrength of a signal from a labeled component of a sample bound toimmobilized compounds can provide a value for general affinity. If aterm used to define binding strength is referred to as “apparent” whatis meant is a measured value without regard to multivalent binding. Forexample, the measured value of an association constant under conditionsof multivalent binding includes a plurality of effects due to monovalentbinding, among other factors. Unless otherwise specified, bindingstrength can refer to any of these measures referred to above.

The term “nucleic acids” includes any and all forms of alternativenucleic acid containing modified bases, sugars, and backbones includingpeptide nucleic acids and aptamers, optionally, with stem loopstructures.

The term “polypeptide” is used interchangeably with “peptide” and in itsbroadest sense to refer to a sequence of subunit natural amino acids,amino acid analogs including unnatural amino acids. Peptides includepolymers of amino acids having the formula H₂NCHRCOOH (α-amino acids),the formula H₂NCHRCHRCOOH (β-amino acids) and/or analog amino acidshaving the formula HRNCH₂COOH. The subunits are linked by peptide bonds(i.e., amide bonds), except as noted. Often all subunits are connectedby peptide bonds. The polypeptides may be naturally occurring, processedforms of naturally occurring polypeptides (such as by enzymaticdigestion), chemically synthesized or recombinantly expressed.Preferably, the polypeptides are chemically synthesized using standardtechniques. The polypeptides may comprise D-amino acids (which areresistant to L-amino acid-specific proteases), a combination of D- andL-amino acids, beta amino acids, and various other “designer” aminoacids (e.g., beta-methyl amino acids, Cα-methyl amino acids, andNα-methyl amino acids) to convey special properties. Synthetic aminoacids include ornithine for lysine, and norleucine for leucine orisoleucine. Hundreds of different amino acid analogs are commerciallyavailable from e.g., PepTech Corp., MA. In general, unnatural aminoacids have the same basic chemical structure as a naturally occurringamino acid, i.e., an a carbon that is bound to a hydrogen, a carboxylgroup, an amino group, and an R group.

In addition, polypeptides can have non-peptide bonds, such asN-methylated bonds (—N(CH₃)—CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—),ketomethylen bonds (—CO—CH₂—), aza bonds (—NH—N(R)—CO—), wherein R isany alkyl, e.g., methyl, carba bonds (—CH₂—NH—), hydroxyethylene bonds(—CH(OH)—CH₂—), thioamide bonds (—CS—NH—), olefinic double bonds(—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH₂—CO—), wherein R is the “normal” side chain. Thesemodifications can occur at any of the bonds along the peptide chain andeven at several (2-3) at the same time. For example, a peptide caninclude an ester bond. A polypeptide can also incorporate a reducedpeptide bond, i.e., R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acidresidues or sequences. A reduced peptide bond may be introduced as adipeptide subunit. Such a polypeptide would be resistant to proteaseactivity, and would possess an extended half-live in vivo. The compoundscan also be peptoids (N-substituted glycines), in which the sidechainsare appended to nitrogen atoms along the molecule's backbone, ratherthan to the α-carbons, as in amino acids.

The term “polysaccharide” means any polymer (homopolymer orheteropolymer) made of subunit monosaccharides, oligimers or modifiedmonosaccharides. The linkages between sugars can include acetal linkages(glycosidic bonds), ester linkages (including phosphodiester linkages),amide linkages, and ether linkages.

General

The invention provides arrays of compounds, and methods of analyzing thearrays, for use in profiling samples. The arrays include compoundsbinding to components of the samples at relatively low affinities.Although practice of the invention is not dependent on an understandingof mechanism, it is believed that under conditions of monovalentbinding, different degrees of specific binding might be difficult todistinguish from each other and from nonspecific binding. However, theaffinity of compounds binding to components of the samples can beincreased by forming arrays such that components of the samples (e.g.,antibodies or cells) can bind to more than one molecule of a compound atthe same time or through avidity interactions with high densities of thecompounds on the surface. When a sample is applied to an array undersuch conditions, the compounds of the array bind to component(s) of thesample with significantly different affinities generating a profilecharacteristic of the sample. Such a profile usually includes somecompounds having no specific binding to components of the sample andother compounds having different degrees of specific binding tocomponents of the sample. Although such binding interactions arespecific in the sense that overall binding profiles of an array arereproducible for replicates of the same sample and distinguishablebetween different samples, they are not necessarily unique in thatcompounds in the array usually show specific binding albeit of differentdegrees to a number of different components of a sample or differentsamples.

The affinity or apparent affinity of informative compounds (i.e., thoseshowing distinguishable binding to different targets) in an array can bemeasured for monoclonal antibody samples. When measured againstmonoclonal antibodies that do not bind to a selected target, informativecompounds in some arrays often show apparent affinity associationconstants in a range of from about 10⁴ to about 10⁹, from about 10⁶ toabout 10⁹, from about 10⁴ to about 10⁷, or from about 10⁴ to about 10⁶.Association constants of such informative compounds are often within arange of from about 10³ to about 10⁶, or from about 10⁴ to about 10⁵.When measured against a complex sample, similar ranges of apparent oractual association constants are observed; however, in this case, theconstants are a composite of values for multiple different componentswithin a sample binding to the same compound. Such affinities can bedistinguished from nonspecific interactions. The proportion ofinformative compounds (i.e., compounds that show distinguishable bindingamong different targets) can vary depending on the composition of thearray and the sample, but ranges of from about 0.1 to about 50%, fromabout 1 to about 30%, from about 0.1 to about 20%, from about 1 to about15%, or from about 3 to about 12% provide some guide. Given thatmonoclonal antibodies each have their own signature consisting ofbinding to many compounds on the surface, it might have seemedimpossible to meaningfully resolve patient serum samples which maycontain 10⁸ or more distinct antibodies with different bindingspecificities. When an array is hybridized against a complex sample,such as from a patient or subject, the binding profile represents theaggregate effect of multiple components of a sample. Surprisinglydespite the complexity of the samples, different samples are associatedwith different binding profiles. Also surprisingly, the intensity ofbinding profile often differs between patients with a disease or at riskof disease relative to normal patients.

The binding profile of such an array to a sample can be used tocharacterize a sample. For example, the binding profile can be comparedwith binding profiles known to be associated with different diseases orstages of diseases or lack of diseases. Alternatively or additionally,the binding can be analyzed, for example, by using a compound bindingrelatively strongly to a component of the sample to affinity purify anantibody from the sample, or by comparing the sequence of a peptide inthe array known to bind strongly to a component of a sample with aprotein database to identify a protein in the sample. Remarkably, thesame array can generate different and informative profiles with manydifferent samples representing different disease states, disease stages,lack of disease and the like. Moreover, a profile characteristic ofdisease or departure from a non-disease state can be detected very earlyin development of a disease before typical analytical markers of diseasewould be detectable by conventional methods, such as ELISA.

Immunosignaturing

The present application provides methods for improved quality control(QC) of arrays in the form of imumunosignaturing.

In the process of immunosignaturing (see, e.g., Stafford and Johnson“Microarray technology displays the complexities of the humoral immuneresponse,” Exp. Rev. Mol. Diagn. 11:5-8 (2011)), a sample of blood isapplied to a large number of peptides or other molecular heteropolymerseach associated with a feature on a surface (for example, an array offeatures each having a different peptide or group of peptides associatedwith it). The antibodies in the blood bind differentially to themolecular species in each of the features. This forms a pattern ofbinding that provides a detailed insight into the molecular recognitionprofile of the antibodies in the blood. The concept is that any changein health is likely to be represented by a change in this molecularrecognition profile.

The patterned molecular surfaces that measure the molecular recognitionprofile can be created in a number of different ways. It is useful tohave methods of determining the quality of these surfaces aftermanufacturing. The present inventors developed methods that areparticularly useful if the immunosignaturing data from the array itselfserves as the measure of the surface quality of the array.

Overall Correlation Coefficient

The vast majority of the antibody signals on the immunosignature arraysdo not change substantially between healthy individuals and individualswith a particular disease. Thus, in most cases, the correlationcoefficient (or any other measure of overall correlation) betweendifferent peptide arrays is generally high and does not change greatlywith the identity of the blood sample. The present inventors identifiedfor the first time that one approach to assessing the quality of anarray is to compare correlation coefficient values (or some othernumerical evaluation of correlation between samples) and set aparticular range for an acceptable correlation.

Another variation of this approach is to ask directly how many of thepeptides or molecular species show similar or different binding betweenthe array in question and a reference array. This can be done by adirect comparison, peptide by peptide, looking for the number ofpeptides that are substantially different between arrays. This can bedone, for example, by having generated data from enough reference arraysto have determined the standard deviation for each peptide. One can thenask where each peptide in the array in question ranks relative to theaverage and standard deviation. The number of peptides within 1 or 2standard deviations can be used as an indication of how well correlatedthe intensities in the arrays are.

Relating Array Binding Intensity Pattern to Fabrication Sequence

Generally speaking, the arrays used for immunosignaturing are made usingdifferent heteropolymers or sets of molecules creating by linking acommon set of monomers together in a specific order or chemical linkagepattern.

One can represent any such library of molecules in terms of a type ofmonomer at each position in the heteropolymer. For example, one can talkabout a peptide in terms of the position in the amino acid sequence andthe identity of the amino acid placed there.

For relatively large libraries, it becomes possible to ask the question:did a particular monomer in a particular position have the expectedeffect on average binding to the array? Thus, one can average theintensity for every heteropolymer in which position i is occupied bymonomer j and get a value. If the number of peptides is large enough,that value will be statistically different from the average value of theintensity for all heteropolymers on the array and contains informationabout the effect of a particular type of monomer at a particularposition. In the case of in situ fabricated arrays, this oftencorresponds to a fabrication step.

For example, the peptides with an alanine at position 7 in the peptidemay all derive from the same fabrication step. If that step failed, thenit will either have no effect (the peptides containing that amino acidat that position in their theoretical sequence will no longer have astatistically detectable effect) or the nature and magnitude of theeffect may change from the empirically determined effect. In general,the intensities of the peptides or heteropolymers to which a particularmonomer is added by a particular cycle of a synthetic process can beaveraged, and compared to the average value of all peptides orheteropolymers in order to determine whether that particular monomer isstatistically different from all peptides or heteropolymers, and if so,what the new direction and magnitude is.

Non-limiting examples of statistical methods that can be performed todetermine a binding intensity of a sample to an array of the inventioninclude: a) analysis of variance (ANOVA); b) chi-squared test; c) factoranalysis; d) Mann-Whitney U analysis; e) mean square weighted deviation(MSWD); f) Pearson product-moment correlation coefficient; g) regressionanalysis; h) spearman's rank correlation coefficient; i) student'st-test; j) time series analysis; k) Kolmogorov-Smirnov test; l)likelihood analysis; and m) Mixture models.

Compounds for Use in Arrays

Many different classes of compounds or combinations of classes ofcompounds can be used for the arrays and methods of the invention.Classes of compounds include nucleic acids and their analogs,polypeptides (broadly defined as above), polysaccharides, organiccompounds, inorganic compounds, polymers, lipids, and combinationsthereof. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes).The test compounds can be natural or synthetic. The compounds can benaturally occurring or non-naturally occurring. Many different classesof compounds other than nucleic acids can be used, but optionally if thecompounds are nucleic acids, the sample components detected are notnucleic acids. In some arrays, the test compounds have a molecularweight of between about 500 and about 10,000 Da, and optionally betweenabout 1000 to about 4000 Da.

The number of compounds used is a balance between two factors. The morecompounds, the more likely an array will include members havingdetectable affinity for any target of interest. However, a larger numberof compounds also increases the cost of synthesizing and analyzing anarray. Arrays typically have at least 100 compounds. Arrays havingbetween 500 and 500,000 compounds provide a compromise betweenlikelihood of obtaining compounds with detectable binding to any targetof interest and ease of synthesis and analysis. Arrays having, forexample, 100 to 500,000 members or 500-500,000, or 1000-250,000 memberscan also be used. Arrays having, for example, between 10,000 and100,000, between 25,000 and 500,000 or between 50,000 and 350,000 arealso contemplated within the disclosures herein. Alternatively, arrayshaving much larger numbers of members for example, 10²-10⁷ or 1000 to5,000,000 or 500,000 to 2,000,000 can also be used. Such arraystypically represent only a very small proportion of total structuralspace, for example less than 10⁻⁶, 10⁻¹⁰, or 10⁻¹⁵ in the case ofpeptides.

Sequence space means the total number of permutations of sequence of agiven set of monomers. For example, for the set of 20 natural aminoacids there are 20^(n) permutations, where n is the length of a peptide.Although it is widely assumed that most if not all of the residues in apeptide epitope participate in binding to the a target, it is much morelikely that between two and five residues in a 10-12 mer epitope areinvolved in energetically favorable interactions with the target, theother residues are simply there to adjust the positions of the importantresidues, and to prevent inhibition of binding. Therefore, a relativelysmall fraction of the total number of possible peptides can provide agood representation of total sequence space, and include members capableof specific, lower affinity interactions with a wide variety of targets.For example, 500-500,000 random peptides can sample the shape space ofan immune system (10⁷ to 10⁸ antibodies in humans) well enough todistinguish between patients with a disease and patients without.

More compounds in the array should allow higher resolution of thediversity of compounds in the complex sample. For example, an array of 1million compounds should allow more resolution of complex samples,including reflecting the complexity of antibodies in a subject's sample.Yet, even with a much smaller number of compounds, one is able to detectand identify immune responses from infection or immunization.

For polymeric compounds, the lengths of polymers represent a compromisebetween binding affinity and ease of synthesis. Length of peptides canaffect both the affinity and specificity of binding. However, as peptidelength increases the chances that any particular binding event willutilize the entire peptide sequence effectively decreases. Cost ofsynthesis also increases with increasing length while fidelity ofsynthesis generally decreases. For peptide arrays, peptides having 4-35,12-35, 15-25 or 9-20 residues are preferred. These ranges of monomerlengths can also be used for other polymers, although aptamers usuallyhave longer lengths (e.g., up to 100 nucleotides).

The compounds (e.g., all or at least 80, 90 or 95%) are typically chosenwithout regard to the identity of a particular target or naturalligand(s) to the target. In other words, the composition of an array istypically not chosen because of a priori knowledge that particularcompounds bind to a particular target or have significant sequenceidentity either with the target or known ligands thereto. A sequenceidentity between a peptide and a natural sequence (e.g., a target orligand) is considered significant if at least 30% of the residues in thepeptide are identical to corresponding residues in the natural sequencewhen maximally aligned as measured using a BLAST or BLAST 2.0 sequencecomparison algorithm with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web sitencbi.nlm.nih.gov/BLAST or the like). Because no particular target isused in the design of the arrays (the arrays are designed to broadlycover the space of molecular recognition), essentially any target willbind to the array and form a signature.

Some compounds are randomly selected from total sequence space or aportion thereof (e.g., peptides in which certain amino acids are absentor under-represented). Random selection can be completely random inwhich case any compound has an equal chance of being selected fromsequence space or partially random in which case the selection involvesrandom choices but is biased toward or against certain monomers, such asamino acids. Random selection of peptides can be made for example by apseudorandom computer algorithm. The randomization process can bedesigned such that different amino acids are equally represented in theresulting peptides, or occur in proportions representing those innature, or in any desired proportions.

In some instances, cysteine residues are omitted from library memberswith the possible exception of a terminal amino acid, which provides apoint of attachment to a support. In some libraries, certain amino acidsare held constant in all peptides. For example, in some libraries, thethree C-terminal amino acids are glycine, serine and cysteine withcysteine being the final amino acid at the C-terminus. A library chosenby random selection, once selected is of known identity and can bereproduced without repeating the initial random selection process.Nevertheless, the compounds in such library retain the same randomrelations with one another. For example, the peptides in a randomlibrary that is subsequently reproduced retain a random distributionthroughout sequence space (with the possible exception of cysteineresidues, if this residue is omitted). Collections of compounds, such aspeptides, that are randomly distributed over sequence space, are stillconsidered random even if reproduced without repeating the initialrandom selection.

The principles for selecting peptides and other compounds for arrays inthe present methods are analogous to those for selecting initiallibraries of compounds in producing synthetic antibodies, as furtherdescribed in WO/2008/048970 and WO2009/140039, the disclosures of whichare incorporated by reference in its entirety herein.

Making Arrays

Compounds can be pre-synthesized and spotted onto a surface of an arrayor can be synthesized in situ on an array surface (see, e.g., Cretich etal., Biomol. Eng. 2, 77-88 (2006); Min et al., Current Opinion inChemical Biology 8, 554-558 (2004), Breitling, Mol. BioSyst., 5, 224-234(2009), U.S. Pat. No. 5,143,854; EP 476,014, Fodor et al., 1993, Nature364, 555-556; U.S. Pat. Nos. 5,571,639, 5,593,839, EP 624,059, U.S. Pat.No. 6,620,584, and EP 728,520). Customized arrays are also commerciallyavailable from suppliers such as Invitrogen, PEPperPRINT, LC Sciences orPepscan. The surface is usually derivatized with a functional group thatattaches to the compounds, optionally via linker. Compounds can beattached via covalent or noncovalent linkages. The array surface can bea single contiguous surface of a support.

Alternatively an array can be formed by spotting or synthesizingdifferent compounds on different particulate supports, such as beads.Peptides can be attached in either orientation (N or C) relative to thearray. In general, the different compounds occupy different areas of acontiguous array or different particles in a particulate array. Theidentity of which compound occupies which area of an array or whichparticle is usually either known as a result of the synthesis process ordeterminable as a result of an encoding process. Encoding processes arecommonly used for beads. The different areas in a contiguous array canbe immediately adjoining as may arise when such arrays are the result ofin situ synthesis, or separated, which is often the result of spotting.

An area or cell of an array is a unit of surface area from which aseparate signal is detectable. In some arrays, each area of the array isoccupied only by molecules of the same compound except for possibly asmall degree of bleed over from one area to another, due for example, toimperfections in the array. In other arrays, some or all of the areascontain a pool of two or more different compounds. In such an array, thesignal from an area containing a pool of two or more different compoundsis the aggregate undivided signal from the compounds constituting thepool.

Such arrays typically contain from 100-5,000,000 compounds (e.g.,100-1,000,000, 500, 100,000 or 500-25,000 compounds) as discussed above.These numbers of compounds can readily be accommodated in differentregions of an array of the order of 0.1-5 cm² combined area.

Within any one area of a contiguous array or within any one particle ofa particle array many different molecules of the same compound arepresent. Because compounds are usually attached to a derivatized surfaceof a support or particle (e.g., a support or particle bearing a linker),the density of molecules within an area of an array or a particle can becontrolled in part by the derivatization process, for example, theperiod of time and concentration of derivatizing agent used. The densityof molecules can also be controlled by the attachment or in situsynthesis process by which a compound is attached to a support. Thelength of a coupling cycle and concentration of compound used incoupling can both affect compound density.

The density of different molecules of a compound within an area of anarray or on a particle controls the average spacing between molecules ofa compound (or compounds in the case of a pooled array), which in turndetermines whether a compound is able to form enhanced apparent affinityto a sample (an avidity interaction). If two molecules of a compound orcompounds in the case of a pooled array, are sufficiently proximate toone another, both molecules can enhance apparent affinity to the samebinding partner. For peptides of length 15-25 residues an average (mean)spacing of less than 0.1-6 nm, 1-4 nm, 2-4 nm, e.g., 1, 2 or 3 nm is,for example, suitable to allow different regions of the same compound toundergo binding with enhanced apparent affinity. Average (e.g., mean)spacings are typically less than 6 nm because spacings of 6 nm or moreare do not allow avidity to enhance the apparent affinity for the targetor cooperative binding to take place. For example, for peptides oflengths 15-25 residues, the two identical binding sites of one antibodycould not span more than 6 nm to contact two peptides at once and bindcooperatively. The optimum spacing for enhancing avidity and/orcooperativity interactions may vary depending on the compounds used andthe components of the sample being analyzed.

Enhancement of apparent affinity through either cooperative binding oravidity interactions can be shown by several methods, includingcomparing binding strength of an antibody to an otherwise identicalantibody fragment (e.g., a Fab fragment) having only one binding site.Binding strength to the intact antibody that is greater than theantibody fragment (e.g., higher apparent association constant) maydifferentiate cooperativity from enhanced avidity. Enhancement ofbinding strength can also be shown by comparing the binding of an arrayof an immobilized compounds to an intact antibody with two binding siteswith the reverse format in which the antibody is immobilized and thecompound is in solution. Stronger binding (e.g., higher apparentassociation constant) of the immobilized compound to the antibody insolution compared with immobilized antibody to the compound in solutionprovides an indication that the immobilized compound can either formmultivalent bonds to the antibody (cooperative binding), or interactsvia enhanced avidity. Association constants, or apparent associationconstants, of compounds can be measured by conventional methods usingtechnologies like SPR, ELISA, Luminex and other solution-phase binding(e.g., monitoring changes in bound signal over time) when the antibodyor other sample is immobilized and the compound is in solution.Conversely, apparent association constants can be measured when acompound is immobilized and antibody or other sample is in solution.Once suitable synthesis or deposit conditions have been established forachieving arrays capable of enhanced binding, other arrays can be madeunder the same conditions without individualized testing.

Usually, different compounds are deposited or synthesized in differentareas of an array under the same conditions, so that if one compound isspaced so that it is capable of enhanced avidity binding, most or allcompounds are. In some arrays, at least 10%, 50%, 75%, 90% or 100% ofcompounds in the array are spaced so as to permit enhanced avidityinteractions and/or undergo cooperative binding with a binding partner.However, it is not necessary that all compounds be deposited orsynthesized with the same spacing of molecules within an area of thearray. For example, in some arrays, some compounds are spaced furtherapart so as not to permit or permit only reduced avidity interactions orcooperative binding compared with other compounds in an array.

The spacing can be measured experimentally under given conditions ofdeposition by depositing fluorescently labeled compounds and countingphotons emitted from an area of an array. The number of photons can berelated to the number of molecules of fluorescein in such an area and inturn the number of molecules of compound bearing the label (see, e.g.,U.S. Pat. No. 5,143,854). Alternatively, the spacing can be determinedby calculation taking into account the number of molecules depositedwithin an area of an array, coupling efficiency and maximum density offunctional groups, if any, to which compounds are being attached. Thespacing can also be determined by electron microscopy of an array or viamethods sensitive to the composition of molecules on a surface such asx-ray photoelectron spectroscopy or secondary ion mass spectrometry.

Arrays having larger spacing that do not permit cooperative binding oravidity interactions or do so to a reduced extent compared with spacingdescribed above also have application in identifying high affinityinteractions. This type of strategy can be used to identify peptides orother compounds, for example, that are very close structurally to theoriginal epitope that raised the antibody response. Alternatively, forarrays of peptides from life space (the set of amino acid sequencesrepresented in the proteins of living organisms), this spacingfacilitates identifying the true epitope.

The spacing between compounds can also be controlled using spacedarrays; that is, arrays on surfaces coated with nano-structures thatresult in more uniform spacing between compounds in an array. Forexample, NSB Postech amine slides coated with trillions of NanoConeapexes functionalized with primary amino groups spaced at 3-4 nm for adensity of 0.05-0.06 per nm² can be used.

Array formats that can be used include microarrays, beads, columns,dipsticks optical fibers, nitrocellulose, nylon, glass, quartz, mica,diazotized membranes (paper or nylon), silicon, silicones,polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals,metalloids, semiconductive materials, quantum dots, coated beads, otherchromatographic materials, magnetic particles; plastics and otherorganic polymers such as polyethylene, polypropylene, and polystyrene;conducting polymers such as polypyrole and polyindole; micro ornanostructured surfaces, nanotube, nanowire, or nanoparticulatedecorated surfaces; or porous surfaces or gels such as methacrylates,acrylamides, sugar polymers, cellulose, silicates, and other fibrous orstranded polymers.

An exemplary method of array preparation is as follows. A microarray isprepared by robotically spotting distinct polypeptides on a glass slidehaving an aminosilane functionalized surface. Each polypeptide has aC-terminal glycine-serine-cysteine as the three C-terminal residues andthe remaining (17) residues determined by a pseudorandom computationalprocess in which each of the 20 naturally occurring amino acids exceptcysteine had an equal probability of being chosen at each position.Polypeptides are conjugated to the aminosilane surface by thiolattachment of the C-terminal cysteine of the polypeptide to a maleimide(sulfo-SMCC, sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate which is covalentlybonded to the aminosilane surface. The polypeptides are chemicallysynthesized, dissolved in dimethyl formamide at a concentration that mayrange from about 0.1 mg/ml to about 2 mg/ml, and then diluted 4:1 withphosphate-buffered saline prior to spotting. The concentration ofpeptide or other compound determines the average spacing between peptidemolecules within a region of the array. A concentration of 1 mg/ml givesan average spacing of about 0.5 nm. The spacing decreases non-linearlywith dilution at lower concentrations. The printed slides stored underan argon atmosphere at 4° C. until use.

An exemplary calculation of spacing is as follows: spot size: 150 μm,spot area: 17671 μm², nanoprint deposition volume: 200 pL, peptideconcentration: 1 mg/ml, deposition amount: 200 pg, #peptides deposited:8×10¹⁰ per spot, 8×10¹⁰ peptides/17671 μm²=4.5×10⁶ peptides/μm²,2.2×10⁻⁷ μm² area needed by 1 peptide (4.6×10⁻⁴ μm spacing).

As well as including compounds randomly or without regard to the samplebeing analyzed, arrays can include other compounds known to bindparticular targets, such as proteins, in a sample. These compounds canbe antibodies, synbodies or peptides among others. Usually, suchinteractions are high affinity (e.g., greater than 10⁷, 10⁸ or 10⁹ M⁻¹).The number of such known binding partner compounds can be large, forexample, there can be a different compound for at least 25, 50, 75, or90% or substantially all of the known proteins expressed by a givengenome, such as the human genome). The different known binding partnercompounds occupy different areas of the array in similar fashion torandomly selected compounds. However, because the known binding partnercompounds are in general capable of high affinity interactions, they canbe used with or without an intermolecular spacing that permits enhancedavidity interactions with the sample. Although one might think thatinclusion of compounds selected at random or without regard to thesample being analyzed would be redundant in view of inclusion of knownbinding proteins to a large part or all of the encoded proteins in agenome, such is not the case because some diagnostic immune responsesare the result of somatic mutation or non-protein components and notdetected by binding proteins to encoded proteins.

Samples and Components to be Analyzed

The arrays and methods of the invention can be used for analyzing anykind of sample containing or potentially containing analyte(s) ofinterest. Of particular interest are samples from human or veterinarypatients or laboratory model animals. Such samples can be blood(including whole blood, red cells, plasma and the like), urine, feces,saliva, CNS fluid, other body fluids, hair, skin, biopsies and the like.A profile can be obtained from a small volume of sample, e.g., lessthan, or equal to, for example, 1 μl. Some samples are from patientsknown or suspected to be suffering from a disease. The identity of thedisease may or may not be known. Some samples are obtained from patientsknown to have been subjected to a risk of disease but in which symptomsof disease are not yet evident. The risk can be genetic (e.g., aparticular gene or family history) or experiential (e.g., exposure to atoxic chemical or radiation). Samples can also be obtained from patientswho have been vaccinated to analyze the resulting immune response.

Samples from patients can include a wide variety of components subjectto potential analysis by an array. The components most amenable todetection are those capable of enhanced avidity interactions tocompounds in the array. Such components include antibodies, which cansupport avidity interactions and/or support cooperative binding throughtheir pairs of heavy and light chains (i.e., two binding sites perantibody) and cells, which can form multiple bonds through multiplecopies of receptors displayed from their outer surfaces. Viruses canalso form enhanced binding interactions through different copies of coatproteins on their outer surface. Samples from patients can include manydifferent antibodies and/or different cells and/or other components.

Samples can be analyzed with little if any further processing or can besubject to further processing such that only selected components of thesample (e.g., antibodies or cells) are analyzed with the array.

Methods of Detection

Binding interactions between components of a sample and an array can bedetected in a variety of formats. In some formats, components of thesamples are labeled. The label can be a radioisotype or dye amongothers. The label can be supplied either by administering the label to apatient before obtaining a sample or by linking the label to the sampleor selective component(s) thereof.

Binding interactions can also be detected using a secondary detectionreagent, such as an antibody. For example, binding of antibodies in asample to an array can be detected using a secondary antibody specificfor the isotype of an antibody (e.g., IgG (including any of thesubtypes, such as IgG1, IgG2, IgG3 and IgG4), IgA, IgM). The secondaryantibody is usually labeled and can bind to all antibodies in the samplebeing analyzed of a particular isotype. Different secondary antibodiescan be used having different isotype specificities. Although there isoften substantial overlap in compounds bound by antibodies of differentisotypes in the same sample, there are also differences in profile.

Binding interactions can also be detected using label-free methods, suchas surface plasmon resonance (SPR) and mass spectrometry. SPR canprovide a measure of dissociation constants, and dissociation rates. TheA-100 Biocore/GE instrument, for example, is suitable for this type ofanalysis. FLEXchips can be used to analyze up to 400 binding reactionson the same support.

Optionally, binding interactions between component(s) of a sample andthe array can be detected in a competition format. A difference in thebinding profile of an array to a sample in the presence versus absenceof a competitive inhibitor of binding can be useful in characterizingthe sample. The competitive inhibitor can be for example, a knownprotein associated with a disease condition, such as pathogen orantibody to a pathogen. A reduction in binding of member(s) of the arrayto a sample in the presence of such a competitor provides an indicationthat the pathogen is present.

The stringency can be adjusted by varying the salts, ionic strength,organic solvent content and temperature at which library members arecontacted with the target.

Applications

The arrays have a wide variety of applications in analyzing orcharacterizing clinical, veterinary, forensic, laboratory and othersamples. As with conventional diagnostics, the arrays can be used toidentify particular analytes within samples, for example, analytesassociated with particular disease. However, the methods can also beused to provide a binding profile of different compounds characterizinga sample. The binding profile represents the aggregate interactions ofthe compounds with different components in the sample, and can becharacteristic of a particular disease, stage of disease or lack ofdisease. The different components can be complex (e.g., at least 10,100, 1000 or 1,000,000,000 different antibodies and/or different cells).

A binding profile typically includes compounds whose interactions withthe sample are nonspecific as well as compounds whose interaction withthe sample reflect specific but low affinity interactions (i.e.,apparent or actual dissociation constant between 10⁻³ and 10⁻⁶).Compounds with higher affinity interactions (i.e., dissociation constantless than 10⁻⁶) may or may not be present. Such higher affinityinteractions if present may arise by chance as a result of a compound inthe array being a mimetic of a natural binding partner of a samplecomponent or as a result of including a control in which a compound is aknown binding partner of a component of a sample. However, a sample canusually be adequately characterized by the binding profile of compoundswith low affinity interactions with the sample, optionally incombination with compounds lacking specific binding to components of thesample. For example, the identity and relative binding of at least 2, 5,10 or 50 compounds capable of low affinity specific binding tocomponents of the sample can often be used to characterize the sample.Such low affinities actions may in part be the result of compoundsserving as mimetopes providing a linear epitope that (imperfectly)resemble an epitope against which an antibody in the same was raised(e.g., a complex 3D-structure).

One application lies in analyzing samples from patients known orsuspected to be suffering from a disease but in which the particulardisease affecting the patient is not known. A conventional approachwould be to perform separate assays for suspected diseases. By contrast,in the present methods, a single binding profile from the patient samplecan be used to characterize the patient for many diseases, stage ofdisease or lack of disease. The binding profile can be used tocharacterize the sample for virtually any disease, including autoimmunedisease, cancer, infectious diseases, and diseases of the CNS. Most ifnot all diseases involve some change s in antibodies, cells or othercomponents present in patient samples, reflected in a binding profile.Some exemplary infectious diseases include bacterial, fungal and viraldiseases, such as Valley Fever, Q-fever, Tularemia tularensis,Rickettsia rickettsii, HSV types I and II, HVB, HVC, CMV, Epstein Barrvirus, JC virus, influenza, A, B or C, adenovirus, and HIV. Becausedifferent infections give different profiles, different infections in apatient having multiple infections can be detected simultaneously. Someexemplary cancers that can be diagnosed or prognosed using the methodsof the invention include glioblastoma, breast cancer, multipleindependent primary cancer and/or recurrence situation, pancreaticcancer, lung cancer, myeloma, ovarian cancer and esophageal cancer.Precancerous cells that are morphological distinguishable from normalcells but not yet cancerous can also be detected using the methods ofthe invention. Neurological diseases, such Alzheimer's disease, althoughnot generally considered to be an autoimmune disease, results in somechanges in antibodies present in a sample. The same is the case forchronic diseases, such as Asthma, Rheumatoid arthritis, Diabetesmellitus type 1, Psoriasis, Multiple Sclerosis and others.

Another application lies in analyzing samples from patients known orsuspected to have a particular disease, but in which the stage, severityor prognosis for the disease is unclear. Again the binding profile canprovide an indication of any of these factors.

Another application lies in analyzing samples from vaccinated patientsto determine whether an adequate protective immune response isdeveloping. The pattern of response in one patient can be compared, forexample, with a patient who has been naturally infected with thepathogen and survived, a similarity of response pattern indicating thepatient is likely to survive and a dissimilarity that the patient willget worse or die at least in the absence of alternate treatment.Alternatively, a profile of a patient or animal model immunized with anew vaccine (for example in a clinical or preclinical trial) can becompared with profiles of patients or control animals immunized with anexisting vaccine known to be effective. In a further variation, patientsbeing recruited for a clinical trial of a vaccine can be prescreened forbinding profile. Those already having a binding profile similar to thatof a patient immunized with a vaccine known to be effective or from apatient who has survived a natural infection can be eliminated from thetrial because their inclusion might lead to a misleading placeboresponse.

Another application lies in screening samples from patients who haveundergone organ transplant (particularly allotransplantation). Theprofile in a patient under test can be compared with profiles ofpatients undergoing organ transplant who have or have not undergonerejection following the transplant. Similarity of the profile between apatient under test and a patient who has previously undergone rejection(or an average profile of a collection of such patients) indicates thatthe patient is at risk or is undergoing rejection.

Another application lies in analyzing samples from a patient known to beat risk of a disease but in which symptoms of disease are not yetpresent. The risk can be genetic, such as a genetic mutation associatedwith disease or family history of the disease, or arise as a result ofexperience, for example, exposure to a toxic chemical, radiation,traumatic accident, stress, fatigue, chemotherapy, unprotected sex, age,or exposure to a subject with a contagious disease. Such a patient isnaturally concerned about the possibility of acquiring a disease andearly therapeutic intervention. The methods are particularly useful incrisis situations in which many subjects have had potential exposure toa risk. Conventional diagnostic assays often have a significant lagperiod before a disease can be developed. For example, conventionalviral assays can take several months to develop detectable patientantibodies. Autoimmune diseases (e.g., lupus, type 1 diabetes,rheumatoid arthritis, multiple sclerosis) can take several years todevelop specific autoantibody or T-cell responses to specificautoantigens. By contrast, the present methods can detect changes in aprofile within a few days (e.g., less than 10, 5 or 3 days) of exposureto a risk, or infection. The changes in binding profile may reflectsubtle changes in concentrations of many different components of asample, few if any of which would be individually detectable. However,in the aggregate, the changes in binding profile of the compounds in thearray indicate a change if the risk has started development of disease.

Another application lies in forensic analysis of a sample, for example,a sample recovered from a crime scene or a sample relevant to apaternity analysis. Comparison of a test sample with one or morereferences samples of known origin can provide an indication of thesource of the test sample.

Binding profiles can be used in a variety of ways in characterizing asample. In some methods, a binding profile of a sample is compared withone or more reference binding profiles of the same compounds. Areference binding profile is a profile that characterizes a particulardisease, stage of disease or lack of disease, and the like. Referenceprofiles are typically determined by averaging binding profiles ofseveral samples (e.g., at least 2, 20, 50 or 100) each characterized forthe same disease, stage of disease or lack of disease. Comparison of asample binding profile with a reference binding profile can involvecomparing the different binding strengths of different compounds in anarray to the respective samples to derive a value representing theoverall similarity of the profiles. A measure of similarity on a scaleof similarity is by implication an inverse measure of disimilarity andvice versa. Thus, a value representing the overall similarity includes avalue representing the overall disimilarity. However, mathematicallydisimilarity matrices can be handled and analyzed distinctly fromsimilarity matrices. Raw data from the sample being analyzed can ofcourse be normalized before the comparison to eliminate any differencesdue to sample size, processing, concentration and the like, rather thanrelative representation of sample components. Standard ANOVA analysescan also block such nuisance factors, provided such factors areaccounted for in the experimental design.

Various techniques can be used to derive a value based upon thecomparison of a binding profile and a reference binding profile. Aderived value can be used to measure the dissimilarity between thebinding profile and the reference profile and be evaluated using adistance measure such as the Euclidean Distance (ED) metric. The EDmetric is typically used for measuring the distance between two vectorsof “n” elements. According to one implementation, if x=(x1, x2, x3, . .. , xN) and y=(y1, y2, y3, . . . , yN) are two points in EuclideanN-space, then the Euclidean distance between x and j may be computed as:D _(xj)=SquareRoot(Summation((x _(i) −y _(i))²))

The ED metric thus not a correlation (0 to 1), but a measurement ofdissimilarity.

In the context of comparing a binding profile (defined by its bindingvalues for each point in N-dimensional space, where N is the number ofexperimental points (conditions)) with a reference binding profile, a EDmetric can be determined regardless of the complexity, number ofpeptides, or number of patients. Each profile being compared may be seenas a pattern: setting an explicit series of points across time, acrossdilutions, across disease states, across symptoms, etc., and thecomparison described here looks for data that reflects this definedseries of points.

To standardize the difference between binding profiles being compared,the calculated ED measurement may be normalized by dividing by thesquare root of the number of conditions as follows:Distance=|a−b|/square root of N

This is distinct from the aforementioned distance calculation bynormalizing for the total number of conditions. This prevents thedistance calculation from expanding too far given large numbers ofsamples.

Accordingly, calculating the Euclidean distance between two data pointsinvolves computing the square root of the sum of the squares of thedifferences between corresponding values. Because the ED metric is ameasure of dissimilarity, the distance (d) may be converted, whenneeded, to a similarity measure as 1/(1+d). Distance, similarity, anddissimilarity are interchangeable to a certain degree but each is auniquely useful given the calculations being applied. As the distancegets larger, the similarity gets smaller. This renders the original datauseful for looking at differences in a non-biased and geometrical way.The computation is scalable with increasing number of experiments. Infact, the complexity of the pattern is inherently diminished to thecalculation because it is in the denominator and is a square root.

Other distance metrics that can be used include Euclidean Squared,Pearson Correlation, Pearson Squared, Spearman Confidence orCorrelation, Kolmogorov-Smirnov test and other like techniques.

Binding profiles can also be used in various analytical methods tofurther characterize the sample. For example, a compound in the arrayshowing relatively strong binding to the sample (compared with othercompounds in the array) can be used to affinity purify a component ofthe sample. The component can then be further characterized (e.g., bysequencing or immunoreactivity). The identity of the compound may becharacteristic of a disease state (e.g., a pathogen, autoantibody ortumor associated antigen). If the component is not already known to becharacteristic of a disease state, it can be used as a new target fordeveloping therapies or diagnostics against the disease state. Forexample, autoantigens or peptides thereof, can be used in inducingtolerance of autoimmune disease. Alternatively, after washing offunbound cellular components, the cellular components binding to an arraycan be dissociated from the array, fractionated and analyzed in similarfashion. In a further variation, the identity of a compound in the arrayshowing relatively strong binding to a sample can be used to identify aligand of the component bound in the sample, and hence the component inthe sample. For example, if the compounds of the array are peptides, thesequence of a peptide showing relatively strong binding to a sample canbe compared with a database of protein sequences. Comparison can bepairwise between a database sequence and a peptide in the array orbetween a database sequence and a motif or consensus sequence from aplurality of peptides in the array. Sequence similarity to a protein inthe database provides an indication that the protein is a ligand of thecomponent in the sample to which the peptide showed strong binding. Theidentity of a ligand in turn provides at least an indication ofpotential molecules in the sample and in turn disease statescharacterized by such molecules.

The same array can be used in any of the applications described aboveand for virtually any disease or suspected disease state. The same arraymeans either literally the same array, in which case the array may bewashed between different samples, or different copies of an array of thesame composition. The identity of which compounds in the array are mostinformative for a disease or other state being analyzed varies by state.Thus, having identified the most informative compounds for a particulardisease, derivative arrays or other detection devices and kits can bemade that have a reduced number of compounds including the mostinformative compounds. The derivative arrays are sometimes referred toas secondary arrays to distinguish them from primary arrays used ininitial identification of binding compounds and sometimes a samplecomponent bound by these compounds.

A further useful aspect of the present methods is that they can detectnot only increased binding of compounds to cellular components in testsamples relative to a control sample representing an undiseased subject(typically a human) but can also detect decreases. For example, somesample components, particularly antibodies, can be detected to decreasein a test sample, such as a disease or vaccinated sample or any other ofthe samples types mentioned, and other sample components increase.

Quality Control Indicators.

The quality of an array on the invention can be determined based on ananalysis of two independent factors: 1) the consistency of fluorescencesignal between corresponding features in multiple arrays; and 2) thesimilarity of the distribution of fluorescence signals measured over thepopulation of features. Surprisingly, an analysis of these two partiallyindependent factors suffices to determine if an immunosignaturing arrayis of an acceptable quality. To better illustrate how these two factorscan be applied to a quality control analysis consider the following:

The correspondence of the binding intensities of different peptidefeatures in multiple, identical arrays that have been used to analyzesamples from different subjects share a degree of similarity. The levelof correspondence can be measured statistically, for example with aPearson Correlation or as a coefficient of variance. The PearsonCorrelation between array data that is sufficiently similar may be atleast 0.7, at least 0.8, at least 0.9, at least 0.95 or at least 0.99.The correspondence between binding intensities of corresponding featuresin a set of arrays can be detected with fluorescence imaging techniques.To illustrate the concept, in any given human population, the height ofan adult human is a trait that should fall within expected ranges, e.g.between about 4 feet to about 7 feet. If one were to measure the samepeople twice, one would expect that the same people that were measuredas being tall the first time would also be tall in the secondmeasurement. If this is not the case, one would expect that themeasurement was faulty. A correlation of detected binding intensitiesfrom the arrays can indicate if an array contains a desired degree ofbinding intensity similarity. If a desired degree of similarity exists,the array has met one quality control threshold. If the desired degreeof similarity does not exist, the array has not met a quality controlthreshold.

In addition, it is possible to measure the shape of the overalldistribution of binding intensities from many different arrays anddetermine how similar they are. These binding distributions can bedetected with fluorescence imaging techniques by measuring fluorescencefrom a dye conjugated to a probe molecule. To illustrate the concept, inany given human population, the height of an adult human is a trait thatshould fall within expected ranges, e.g. between about 4 feet to about 7feet. However, the distribution of heights is not even over this range;there are very few adults that are 4 feet in height and very few thatare 7 feet. One would expect to always find more or less the samedistribution if one is randomly selecting people from the population andmeasuring their height. If one does not find a similar distribution, onemight suspect that the measurement was faulty. Detection of a bindingintensity that corresponds to a known trait suggests that the array hasmet one quality control threshold. Absence of such binding suggests thatthe array has not met one quality control threshold.

In some embodiments, a quality control analysis of an array of theinvention comprises: a) detecting a binding intensity of a sample to aplurality of features on a peptide array; b) determining a bindingintensity of the sample to each feature on the array; and c)statistically correlating the binding intensities of each feature.

Derivative Analyses

In addition to being useful in themselves for analyses of samples asdiscussed above, the present methods are also useful for determiningderivative compounds and detection devices. In a simple form of suchmethods, a derivative device or other array in constructed containingone or more compounds known to be associated with a given disease,susceptibility to disease or other condition described above, andomission of other compounds from the primary array not found to beinformative for this disease, susceptibility or other condition. In somesuch methods, only a small proportion of the compounds used in a primaryarray (e.g., less than 0.1%, 1% or 5% are retained). In other methods, acomponent of the sample bound by some of the compounds in a primaryarray is identified by any of the approaches discussed in the previoussection. Having identified a component of the sample, one or more knownbinding partners of the component are also identified. The known bindingpartners can be compounds from the primary array, antibodies to thecomponent or other compound, such as a synbody that is known to bind tothe component. The known binding partner(s) can then be used to detectthe sample component to which they are known to being by any otherwiseconventional diagnostic assay. For example, if the known binding partneris an antibody, the assay can be an ELISA, immunoprecipitation,radioimmunoassay or the like. If a plurality of known binding partnersare used, the known binding partners can be immobilized in an arrayformat. The known binding partners can also be incorporated intodiagnostic kits or diagnostic device (e.g., attached to a support). Sucharrays, diagnostic devices and kits can be manufactured by conventionalmeans. Of course, once the known binding partners of a component havebeen identified, it is not necessary to repeat the initial screeningwith the primary array for subsequent manufacture of such arrays,diagnostic devices and kits.

Although the embodiments have been described with reference to thepresently preferred embodiments, various modifications can be madewithout departing from the invention. Unless otherwise apparent from thecontext any step, element, embodiment, feature or aspect of theembodiments can be used with any other.

EXAMPLES Example 1 Overall Correlation Coefficient Assessment

Arrays of peptides were fabricated by in situ synthesis such that therewere 330,000 peptides in each array. Twelve such arrays were subjectedto immunosignaturing with six different kinds of samples. In 6 of thesesamples, one secondary antibody for IgM was used. In 6 of these samples,a different secondary antibody for IgG was used. Using an IgG secondaryantibody accidentally instead of an IgM secondary could represent an“error” in the assay condition that would alter the overall pattern ofbinding observed.

Correlation coefficients were determined between all possible pairs ofarrays (Pearson Correlation). The average correlation coefficientbetween the samples that used the IgM secondary antibody was 0.92+/−0.02(these numbers represent that average and the standard deviation, SD).The correlation coefficient between IgM and IgG samples on average was0.59+/−0.06. Each of the individual samples that “mistakenly” used IgGhad correlation values in comparison with the IgM correct assay thatdiffered by a statistically significant margin. It clearly would havebeen possible to detect the use of the “wrong” secondary antibody hadone array been treated incorrectly relative to the others by thismethod.

Accordingly, a failed process may be indicated if the correlationcoefficient between a given assay and a standard assay is greater than athreshold value. In some embodiments the threshold value may be definedas a percentage of the correlation coefficient between assays that weredone correctly. In this case, to pass QC, the threshold percentageshould be at least under 3%, at least under 4%, at least under 5%, atleast under 6%, at least under 7%, at least under 8%, at least under 9%,at least under 10%, at least under 15%, at least under 20%, at leastunder 25%, at least under 30%, at least under 35%, at least under 40%,at least under 45%, or at least under 50%.

Alternatively, in some embodiments the threshold value may be defined interms of the standard deviation (SD) derived from generating manycorrelation coefficients between assays that were done correctly and astandard assay. In this case, to pass QC, the threshold correlationcoefficient between a given assay and the standard assay should be atleast under 2× SD, under 2.5× SD, under 3× SD, under 3.5× SD or under 4×SD.

Example 2 Relating Array Binding Intensity Pattern to FabricationSequence

An array of peptides, with known peptide sequences, was generated by amask-based, in situ synthetic process on a silicon oxide surface. Eachstep in the process consisted of the removal of a blocking group fromthe end of a subset of the growing peptides and addition of a specificamino acid only to those positions until the desired peptide length wasachieved.

The peptide array was then exposed to a sample of human serum and theIgG bound was quantified by addition of a labeled secondary antibody andvisualized. The synthesis of the array deliberately left out the firsthalf of the masks (i.e., those steps were not performed), simulatingfailed steps in the synthesis (masks 1-69). Masks 70-140 were usedproperly to synthesize, and subsequently lengthen, peptides on thearray.

The average intensity values measured for the subset of peptides onlythat should have been modified by each of the synthesis steps above,including synthesis where no masks were used (i.e., simulating that thesteps were not performed) were subsequently determined and plottedagainst each synthesis step. The resulting average values are shown inFIG. 1. As can be seen from FIG. 1, steps in which the synthesis did notoccur, i.e. masks 1-69 where no amino acids were added to the peptidefragment on the array, resulted in a similar, universal average value.This is in stark contrast to the average intensity values obtained forthe subset of peptides affected with masks 70-140, where a statisticallyunique value of binding was obtained for these synthesis steps. Thisstatistically unique pattern of binding for each synthesis step, whichcorresponds with successful synthesis of the peptides on the array, islargely reproducible across different batches of arrays with the samepeptide sequences. In our experience, a unique pattern of binding can beobtained for each synthesis steps that differs by a threshold percentagewhen compared, for example, to a universal average intensity value.

This universal average intensity value can be obtained, for example, byaveraging all of the peptides (e.g., all 330,000 peptides) on the arrayafter synthesis occurs. Other means can be used for obtaining thisuniversal average intensity value, including but not limited toaveraging all peptides on the array after each synthesis step andaveraging the values of those numbers obtained.

An average intensity value at a given synthesis step which does notsignificantly differ from this universal average intensity value mayindicate the failure of synthesis, i.e. addition of an amino acid, atthat step. A failed synthesis step may be indicated if the differencebetween the average intensity value at a given step and the universalaverage intensity value is not above a threshold percentage of about 2%,of about 5%, or about 10%, or about 15%, or about 20%, or about 25%, orabout 30%, or about 35% or about 40%, or about 45%, or about 50%. Thethreshold average intensity value difference in some embodiments shouldbe at least over 2%, at least over 3%, at least over 4%, at least over5%, at least over 6%, at least over 7%, at least over 8%, at least over9%, at least over 10%, at least over 15%, at least over 20%, at leastover 25%, at least over 30%, at least over 35%, at least over 40%, atleast over 45%, or at least over 50%.

Alternatively, a failed synthesis step may be indicated if thedifference between the average intensity value at a given step and theuniversal average intensity value is not above a threshold defined interms of the standard error (SE) determined for the given step's averageintensity. In some embodiments, a failed synthesis step is indicated ifthe difference between the average intensity value at a given synthesisstep and the universal average intensity value is less than 2× SE, lessthan 2.5× SE, less than 3× SE, less than 3.5× SE or less than 4× SE. Insome embodiments, the threshold difference should be at least over 2×SE, at least over 2.5× SE, at least over 3× SE, at least over 3.5× SE orat least over 4× SE.

This pattern of effect is largely reproducible even when differentdiseases are considered, because the vast majority of the bindingsignals from the peptides are not strongly affected (at least relativeto the average). Accordingly, in other embodiments, the determination ofwhether a synthesis step may have failed can be run concurrently withsubject samples as a primary or additional means of quality control ofthe peptide array. Alternatively, this quality control method can alsobe run on single peptide arrays produced in a batch run, either as aprimary or additional means of quality control of the peptide array.

Example 3 Relating Array Binding Intensity Pattern to FabricationSequence

Another way of averaging the data and determining if an amino acidaddition step may have failed during synthesis, is based on the positionand the monomer added. This may be distinct from the step of thesynthesis if a particular step adds a particular monomer at differentpositions within a peptide.

The example shown in Table 1 is for a peptide array produced on asurface. The peptide array consisted of 330,000 peptides. The peptidearray was exposed to a sample of human serum as above, and the IgG boundto the array from the sample was detected with labeled secondaryantibody and visualized. The average value of the intensity of IgGbinding to each peptide that had a particular monomer at a particularposition is determined relative to the average intensity over the wholearray (i.e. all 330,000 peptides), to obtain a relative value of IgGbinding to a particular monomer to a particular position. A matrix ofposition and amino acid was generated. Note that the way the peptideswere made, not all amino acids are present in all positions and thusthere is a blank at position A-13 in the matrix.

One can see that there are considerable variations in average values ofintensity across the matrix because the particular amino acids andpositions have different effects on binding. To determine if thevariations in average values of intensity are due to positional effectsof particular amino acids, the data was re-analyzed without knowledge ofthe particular amino acids and positions. Table 2 is from exactly thesame array as in Table 1, except that the data was scrambled such thatthe correlation between a particular intensity value and a particularpeptide in the array was randomly changed. Note that almost all of thepositions and amino acids now take on the average value of intensity(i.e., everything is normalized to that average value which isconsidered 1.0 for this example). Those positions/amino acids near theedge that show some variation from average do so because the number ofpeptides with that amino acid at that position is small.

TABLE 1 Table 1. Intensity changes for peptide subsets relative toglobal average. The subset of peptides averaged for each valuecorresponds to peptides with the particular amino acid (y-axis) andparticular position in the peptide (x-axis) 1 2 3 4 5 6 7 8 9 10 11 1213 A 0.88 0.91 0.96 1.00 1.05 1.08 1.09 0.98 0.76 0.85 1.06 0.83 D 0.880.96 1.01 1.12 1.15 1.16 1.15 1.15 1.15 1.13 1.12 1.12 1.11 E 0.98 1.091.17 1.16 1.16 1.15 1.16 1.16 1.15 1.14 1.12 1.13 1.12 F 0.77 0.86 0.910.85 0.82 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.85 G 1.17 1.02 1.08 1.071.08 1.07 1.09 1.10 1.12 1.12 1.10 1.10 0.99 H 1.09 1.05 1.09 1.07 1.071.07 1.11 1.17 1.23 1.23 1.23 1.30 1.23 K 0.92 0.94 0.94 0.93 0.95 0.920.93 0.92 0.94 0.93 0.90 0.92 0.95 L 0.90 0.78 0.81 0.82 0.83 0.82 0.800.81 0.84 0.85 0.87 0.91 0.88 N 0.91 0.92 0.95 0.91 0.91 0.90 0.90 0.910.93 0.93 0.93 0.97 0.99 P 1.48 1.22 1.21 1.18 1.18 1.18 1.16 1.16 1.161.16 1.13 1.12 1.16 Q 0.99 1.00 1.01 0.96 0.94 0.94 0.92 0.92 0.91 0.910.90 0.89 0.85 R 0.97 0.93 0.96 0.99 1.00 1.00 1.00 1.02 1.05 1.05 1.041.10 1.07 S 0.93 0.84 0.89 0.91 0.92 0.91 0.92 0.94 0.97 0.98 0.98 0.991.03 V 0.95 0.92 0.92 0.92 0.93 0.93 0.95 0.95 0.97 0.95 0.94 0.93 0.95W 1.31 1.71 1.21 1.24 1.17 1.22 1.19 1.17 1.17 1.13 1.08 1.06 1.03 Y0.88 0.86 0.88 0.87 0.84 0.82 0.81 0.81 0.81 0.81 0.79 0.81 0.78 SD 0.180.22 0.12 0.13 0.12 0.13 0.13 0.14 0.15 0.14 0.13 0.14 0.13 ave 1 1 1 11 1 1 1 1 1 1 1 1

TABLE 2 Table 2. Same as Table 1 except that the assignment of peptidesto the list of intensity values was scrambled. Note that the number ofpeptides in each subset averaged is smaller on the top and right edgesand thus there is greater variance. 1 2 3 4 5 6 7 8 9 10 11 12 13 A 1.001.00 1.00 1.00 1.00 0.99 0.98 0.99 0.88 1.00 0.95 1.09 D 1.01 1.00 1.001.00 0.99 1.00 1.01 1.00 1.00 1.00 1.00 1.00 0.99 E 0.99 1.00 0.99 0.991.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.03 F 1.00 1.00 0.99 0.99 1.021.01 1.00 1.00 1.01 0.99 1.00 0.98 0.95 G 1.00 1.00 1.00 1.00 1.00 1.001.01 1.00 1.01 1.00 0.99 1.00 0.90 H 1.00 1.00 0.99 1.00 1.00 1.00 1.001.00 1.02 1.00 1.01 1.02 1.00 K 0.99 1.01 1.00 1.00 1.00 1.00 1.00 0.991.01 0.99 1.00 1.00 1.01 L 1.01 1.01 1.00 1.01 1.00 1.00 1.00 1.00 1.021.00 0.98 0.99 1.02 N 1.00 1.00 1.00 1.01 1.00 1.00 1.00 1.00 1.00 1.000.97 0.97 0.99 P 1.00 0.99 1.01 0.99 1.00 1.00 1.01 0.99 1.01 1.01 1.031.02 1.04 Q 1.00 1.00 0.99 1.00 1.01 1.00 1.01 1.00 1.01 1.02 1.02 0.961.02 R 0.99 1.00 1.00 1.00 1.00 0.99 1.00 1.00 1.00 0.98 1.00 0.99 0.99S 0.99 1.00 1.00 1.00 0.99 1.00 1.00 1.01 1.01 1.01 1.01 1.01 0.94 V1.00 0.99 1.00 1.00 1.00 1.00 1.01 1.00 1.01 1.00 1.00 0.99 1.04 W 1.000.99 1.03 1.01 1.00 1.00 1.00 1.01 1.01 1.00 1.01 1.00 1.05 Y 1.01 1.001.01 1.00 0.99 1.00 1.00 1.00 1.00 1.00 1.01 1.00 1.04 SD 0.01 0.01 0.010.01 0.01 0.00 0.01 0.01 0.03 0.01 0.02 0.03 0.04 ave 1 1 1 1 1 1 1 1 11 1 1 1

As above, the universal average intensity value can also be obtained,for example, by averaging all peptides on the array after each synthesisstep and averaging the values of those numbers obtained.

A relative intensity value for an amino acid at a particular position ona peptide may indicate the failure of synthesis, i.e. addition of anamino acid, where the synthesis step occurred. A failed synthesis stepmay be indicated if the relatively intensity value obtained is not abovethe universal average intensity value (i.e. 1.0) by a thresholdpercentage of about 2% or about 5%, or about 10%, or about 15%, or about20%, or about 25%, or about 30%, or about 35% or about 40%, or about45%, or about 50%. The threshold average intensity value difference insome embodiments should be at least over 2%, at least over 3%, at leastover 4%, at least over 5%, at least over 6%, at least over 7%, at leastover 8%, at least over 9%, at least over 10%, at least over 15%, atleast over 20%, at least over 25%, at least over 30%, at least over 35%,at least over 40%, at least over 45%, or at least over 50%.

Alternatively, a failed synthesis step may be indicated if therelatively intensity value obtained is not above the universal averageintensity value (i.e. 1.0) by a threshold defined in terms of thestandard error (SE) determined for the relative intensity value. In someembodiments, a failed synthesis step is indicated if the differencebetween the average intensity value at a particular position for a givenamino acid and the universal average intensity value is less than 2× SE,less than 2.5× SE, less than 3× SE, less than 3.5× SE or less than 4×SE. In some embodiments, the threshold difference should be at leastover 2× SE, at least over 2.5× SE, at least over 3× SE, at least over3.5× SE or at least over 4× SE.

As above, the determination of whether a synthesis step may have failedcan be run concurrently with subject samples as a primary or additionalmeans of quality control of the peptide array. Alternatively, thisquality control method can also be run on single peptide arrays producedin a batch run, either as a primary or additional means of qualitycontrol of the peptide array.

While preferred embodiments have been shown and described herein, itwill be obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, andsubstitutions will now occur to those skilled in the art withoutdeparting from the embodiments. It should be understood that variousalternatives to the embodiments described herein may be employed inpracticing the described methods. It is intended that the followingclaims define the scope of the embodiments and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

Example 4 Analytic Framework for Quality Control (QC) Analysis

We describe herein an analytic framework that has been utilized indetermining the quality and integrity of an array of the invention.

An array of the invention was synthesized with ˜350,000 features on acomposite silicon crystal/silicon oxide wafer. The silicon oxide layeris directly functionalized with linker groups suitable for peptidesynthesis. Manufactured arrays with peptides were incubated with a testantibody. This antibody is detected with an anti-idiotype, anti-speciessecondary antibody with a fluorescent tag. The wafers were scanned andconverted to numerical values by applying a virtual grid over the imageand calculating the measured intensities for each of the ˜350,000peptides. This process creates a ‘gpr’ file containing a list of theintensities for each peptide, and the relative location of the peptidesin the image. This text file containing the numerical representation ofthe array image is then used for subsequent numerical analysis. In thepresent example, an Innopsys Innoscan 910AL 2-color laser scanner isused for acquiring the image, and the same acquisition software is alsoused to automatically grid and convert the images. Any laser scannerwith 1.0 micron resolution, 550 nm and 650 nm lasers, and highdiscrimination emission filter is capable of scanning peptide arrayssynthesized on opaque and partially reflective silicon/silicon oxidesurfaces. Images and gpr files are stored in a fileserver until needed.

Each gpr file contains specific columns representing aspects of thetranslated image. One of the columns is denoted “Median ForegroundXXXnm” column. “XXXnm” corresponds to the wavelength of the fluorophorelinked to the detection antibody. In this example, multiple wavelengthswere detected simultaneously, as multiple isotypes can be detected usingthe two colors that the scanner can detect. More than one antibody withdistinct isotypes, such as IgG and IgM, can be used in a quality controlanalysis.

Gpr files are read into R, a free-ware 64-bit analytical/statisticalprogramming language (CRAN.org). A matrix is created from the MedianForeground column, with each row given a unique peptide name, and eachcolumn given a unique sample name. No peptide or sample averaging isperformed.

The content within this matrix represents the data of interest for thequality control analysis. Each quality control analysis can be performedon arrays from multiple wafers. Table 3 illustrates the parametersconsidered by a computer program product in performing a quality controlanalysis of an array.

TABLE 3 sortArray_QC <− # sortArray_QC( ) rank orders immunosignaturingarrays by similarity# QC <−sortArray_QC(FG,arraylimit=10,pepsmpl=20,cor.scale=0.2,ks.scale) #ARGUMENTS: #FG: numeric matrix of foreground spot intensities from animmunosignaturing #array, with peptide features in rows and arrays incolumns. #arraylimit: integer; minimum number of arrays to compare-- thestopping point for the algorithm. #pepsmpl: integer; fraction ofpeptides to sample, where 20 means every 20th; pepsmpl=1 will use theentire set. #cor.scale: number; correlation scaling factor forcalculating distance; default value is 0.20. #ks.scale: number; scalingfactor to apply to mean KS p-values when calculating the QC distance. Ifomitted, it will be calculated as: max(mean.ks) − mean(mean.ks), 1stiteration # VALUE; returns a data.frame with rownames taken from thecolumn names of FG, ordered by the rank column, and columns: #rank: rankorder of the arrays, from best (1) to worst (2) #mean.cor: mean Pearsoncorrelation to arrays with lower rank values #mean.ks: meanlog10(p-value), KS test of distribution, to arrays with lower rankvalues #qc.dist: the distance calculated from scaled mean.cor andmean.ks

Fluorescence values, sample name, peptide number and peptide sequenceenter the QC program. Values returned from the analysis are linear,unscaled distances that integrated two orthogonal measures ofconsistency and reproducibility. First, the Kolmogorov-Schmirnovgoodness of fit (ks.gof) value represents a measure of the similarity ofthe data distribution between two arrays of data. Second, Pearson'sCorrelation Coefficient (cor) represents the linear relatedness betweentwo arrays of data. The distance value is a representation of anear-even weighting of the average of these assessments across alltested arrays. This means every possible comparison across any number ofarrays is performed, and the average for these samples is returned. Afixed cutoff of 0.70 avg. correlation and a value of <2.5 for theavg.−log₁₀ KS-GOF score was used in evaluating a threshold quality levelfor the wafers.

A non-reported value was also calculated, to be used as a checksum thatensures the scores calculated above represent authentic, reproducible,expected data and convey array performance. After the acceptable ks.govand cor values were determined, the coefficient of variation (CV,stdev/mean) is calculated for each peptide across the samples beingtested. The average CV for each array is computed and assigned to avector. As the QC process iterates, files that did not meet theaforementioned minimum threshold were discarded iteratively. Eachiteration discards arrays that failed to meet the minimum threshold forreproducibility. Each iteration, the average CV per array is calculated.As files that do not meet the minimum quality control threshold wereremoved from the list, the average CV across the remaining arrays shoulddecrease, indicating that the program is removing the files that had theworst reproducibility. The CV values are stored in a vector.

CV's are expected to somewhat vary from synthesis-to-synthesis. However,if a downward trend in CV is not observed in the analysis frameworkdescribed above, the original data should be independently examined forpossible image artifacts or biases not detectable by theKS-GOF/Correlation calculation. This set of CV values can then beanalyzed using a Kolmogorov complexity (KC) calculation. If the KC valueexceeds KC>0.49, the images should be examined for systematic bias orimage artifacts, or other parts of the image acquisition, gridding, oralignment process should be considered suspect.

As analysis and manufacturing changes are implemented, it may benecessary to revisit the minimum threshold values currently used todetermine the fabrication of an array of acceptable quality. In theexperiment described herein, the threshold cutoff was decidedempirically through multiple rounds of testing immunosignatures thatcorrespond to various disease states, various arrays, and variousmonoclonal antibodies. The cutoff is presently near the range of humanto human variability, ensuring that the technical variability does notexceed the biological variability.

Example 5 Analytical Analysis Performed After Quality Control ofAcceptable Files

Once an array is found to conform to the Quality Control standardsspecified in Example 4, further quality control analysis can beperformed using the following framework. This framework analysis can beapplied to selection/training, sample prediction of unknowns, leave-outcross-validation, or holdout validation. Selection is the process bywhich peptides are selected to give the best prediction of disease in awell-defined cohort and only in that cohort. Should the cohort be 1disease vs. healthy, the selection process is limited to only detectingthat disease against healthy persons. Should the cohort be 1 disease vs.non-disease, the selection process now encompasses non-healthy butnon-disease 1 samples. Sample prediction of unknowns is the process bywhich the selection/training process is validated. If the trainingprocess was robust, any disease that was never used in the trainingprocess, and is blinded to the analyst, should be detected andclassified as the disease at high accuracy. This test process enablesone to ensure the training process is adequate or needs to be expanded.Leave-out cross-validation is used when blinded samples are unavailable.Cross-validation removes (typically) 1 sample from the dataset, which isthen used as the test data. This process of removing and testing samplesis done repeatedly until every sample was tested once. Holdout is aprocess of removing a fixed percentage of samples from the trainingprocess, and then using them as test samples. This is nearly identicalto a blinded train/test trial, other than the holdout samples are knownto the analyst. There is no mathematical difference between blindedtrials and holdout trials.

The following example describes further steps taken in the qualitycontrol analysis of the array described in Example 4.

Each gpr file can be loaded into GeneSpring 7.3.1 (subsequently referredto as GS, currently sold by Agilent Technologies, Santa Clara, Calif.).GS creates a matrix of columns from the gpr files. One columncorresponds to all ˜350,000 peptides and 1 sample. In this example, amatrix was created in GS that listed each peptide in the array in a rowand the sample analyzed in a column. In the current example, QCanalysis, values for each gpr file of each identical peptide wereaveraged. Values less than 0.01 were converted to 0.01. FIG. 2 is aflowchart illustrating actors and processes used in the imageacquisition, alignment, translation, and analysis described herein.

The overall fluorescence signal was measured and the median signal foreach of the ˜350,000 peptides in the array was taken from the gpr file.The value measured for each feature was divided by the median value thatwas calculated.

Subsequently, the median signal for each of the 350,000 peptides andeach sample being analyzed was calculated. Each value from each arrayfor a given peptide was divided by the median signal for that peptide.

Samples were assigned attributes. Attributes included ‘disease’,‘technical replicate’, ‘biological replicate’, ‘wafer’, ‘assay’, etc.The attributes were used to average replicates; identically-namedsamples (technical replicates) were averaged for further analysis.

Each disease that was analyzed was assigned a disease class. Diseaseclasses were in turn used for subsequent feature selection.

1) Feature selection: Once attributes were assigned and averages weredefined, the feature selection process began. The number of diseaseclasses was defined by the attributes.

2) Statistical analysis: For a two-class analysis, feature-selection wasperformed by t-test between the two samples, then pattern matching.T-test is a Welch-corrected t-test with multiple testing accounted forby setting Family Wise Error Rate to 5% (FWER=5%), reducing falsepositives to 5% of the total features selected. Pattern matching wasperformed in GS by selecting a pattern of expression. The selectedpattern was defined as consistently high for class 1 and consistentlylow for class 2 using Pearson's Correlation coefficient as the metric tojudge the goodness of fit to the selected pattern.

In the analysis described in this example, twenty-five peptides wereselected from the t-test pre-filtered list that survived the patternmatching analysis. The High/Low pattern is then reversed and twenty-fivemore peptides are selected. Fifty peptides were used for two-classprediction.

For multi-class analysis, feature selection was performed by Type IFixed-Effects ANOVA with setting Family Wise Error Rate to 5% (FWER=5%).Pattern matching was done as described in the statistical analysis 2),but for each class, a pattern of UP for that class, and a pattern ofDOWN for all other classes was determined with 50 total peptidesselected for each class.

3) Classification: For experiments analyzing only 2 classes, LDA, kNN,NB and SVM classifiers were determined with Leave One OutCross-Validation. Error for each classifier was provided. Forexperiments using multiple classes, the LDA was not used due topropensity to error due to colinearity affects. In the analysisdescribed in this example, the false positive rate was equal to falsenegative rate.

Within classification, a resampling could be performed given asufficient sample size. Resampling is a process by which samples arerandomly selected to serve as “training” and “test” at a predefinedratio. A suitable resampling ration is a 4:1 ratio of training to test.

4) Validation: either a permuted t-test or permuted ANOVA could beperformed to ensure that there were no group labels that produced lowerp-values than the correct assignments. With 350,000 peptides, poorreproducibility can yield situations where p-values are not in the 10⁻⁸to 10⁻³⁰ range deemed acceptable for an array of the invention.

5) Charting and reports: a classification error was reported as FP, FN,TP, TN rates, NPV, PPV, sensitivity, specificity, and harmonic mean.Charts included 2 dimensional hierarchical clustering using Euclideandistance, Pearson's correlation, or other metric, specifically usingaverage linkage as the metric for creating dendrimer linkages(heatmaps).

PCA charts can provide relative class separation in graphical format.ROC curves can be provided as charts when desired/needed using pROClibrary in S+.

Example 6 Computer Architectures

Various computer architectures are suitable for use with the invention.FIG. 3 is a block diagram illustrating a first example architecture of acomputer system 300 that can be used in connection with exampleembodiments of the present invention. As depicted in FIG. 3, the examplecomputer system can include a processor 302 for processing instructions.Non-limiting examples of processors include: Intel Core i7™ processor,Intel Core i5™ processor, Intel Core i3™ processor, Intel Xeon™processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-Sv1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8Apple A4™ processor, Marvell PXA 930™ processor, or afunctionally-equivalent processor. Multiple threads of execution can beused for parallel processing. In some embodiments, multiple processorsor processors with multiple cores can be used, whether in a singlecomputer system, in a cluster, or distributed across systems over anetwork comprising a plurality of computers, cell phones, and/orpersonal data assistant devices.

Data Acquisition, Processing and Storage.

As illustrated in FIG. 3, a high speed cache 301 can be connected to, orincorporated in, the processor 302 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 302. The processor 302 is connected to a north bridge 306 by aprocessor bus 305. The north bridge 306 is connected to random accessmemory (RAM) 303 by a memory bus 304 and manages access to the RAM 303by the processor 302. The north bridge 306 is also connected to a southbridge 308 by a chipset bus 307. The south bridge 308 is, in turn,connected to a peripheral bus 309. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 309. In some architectures, thefunctionality of the north bridge can be incorporated into the processorinstead of using a separate north bridge chip.

In some embodiments, system 300 can include an accelerator card 312attached to the peripheral bus 309. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing.

Software Interface(s).

Software and data are stored in external storage 313 and can be loadedinto RAM 303 and/or cache 301 for use by the processor. The system 300includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem.

In this example, system 300 also includes network interface cards (NICs)310 and 311 connected to the peripheral bus for providing networkinterfaces to external storage, such as Network Attached Storage (NAS)and other computer systems that can be used for distributed parallelprocessing.

Computer Systems.

FIG. 4 is a diagram showing a network 400 with a plurality of computersystems 402 a, and 402 b, a plurality of cell phones and personal dataassistants 402 c, and Network Attached Storage (NAS) 401 a, and 401 b.In some embodiments, systems 402 a, 402 b, and 402 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 401 a and 402 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 402 a, and 402 b, and cell phone and personal dataassistant systems 402 c. Computer systems 402 a, and 402 b, and cellphone and personal data assistant systems 402 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 401 a and 401 b. FIG. 4 illustrates anexample only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some embodiments, processors can maintain separate memory spaces andtransmit data through network interfaces, back plane, or otherconnectors for parallel processing by other processors. In someembodiments, some or all of the processors can use a shared virtualaddress memory space.

Virtual Systems.

FIG. 5 is a block diagram of a multiprocessor computer system using ashared virtual address memory space. The system includes a plurality ofprocessors 501 a-f that can access a shared memory subsystem 502. Thesystem incorporates a plurality of programmable hardware memoryalgorithm processors (MAPs) 503 a-f in the memory subsystem 502. EachMAP 503 a-f can comprise a memory 504 a-f and one or more fieldprogrammable gate arrays (FPGAs) 505 a-f. The MAP provides aconfigurable functional unit and particular algorithms or portions ofalgorithms can be provided to the FPGAs 505 a-f for processing in closecoordination with a respective processor. In this example, each MAP isglobally accessible by all of the processors for these purposes. In oneconfiguration, each MAP can use Direct Memory Access (DMA) to access anassociated memory 504 a-f, allowing it to execute tasks independentlyof, and asynchronously from, the respective microprocessor 501 a-f. Inthis configuration, a MAP can feed results directly to another MAP forpipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. Any variety of datastorage media can be used in connection with example embodiments,including random access memory, hard drives, flash memory, tape drives,disk arrays, Network Attached Storage (NAS) and other local ordistributed data storage devices and systems.

In example embodiments, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other embodiments, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 5, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 312 illustrated in FIG. 3.

In some embodiments, the quality control analysis described herein wasperformed on an Apple Mac Pro, 64 bit architecture, 1 TB SSD drives, 128G RAM, dual 12 core Xeon CPU's running 3.06 GHz using the Windows 7 OS.The system described in the analyses of Examples 1-6 had an on-linestorage with a dual-Gigabit Ethernet network attached storage (NAS).Internet security for the system described in the analyses of examples1-6 was provided by Oracle 9i.

FIG. 6 is a diagram of single-machine parallel problem solving(multi-core, multi-CPU problem solving). Illustrated here is a diagramof commercially available one-box compute-farm (i.e. Apple Pro).Medium-speed clock coupled with full 64-bit wide memory access,high-speed L1 and L2 cache, integrated parallel GPU processors, andsolid-state storage mean a tuned system with no bottlenecks. Thisdiagram represents a model system capable of coupling to currentdisclosed invention.

Example 7 A Method of Quality Control

Frequently, immunosignature assays are performed in large batches ofseveral hundred assays at a time. A general feature of immunosignaturesis that the effect of the disease is limited to a relatively smallnumber of peptides on the surface. Therefore most peptides behavesimilarly in any two arrays, regardless of the health status of thepatient. Because of this, one can compare individual array-based assaysin large batches for consistency. Assays that give substantiallydifferent overall results from the others are rerun. It is frequentlynecessary to consider at least two types of correlations between assayresults in determining assay quality.

One is the correspondence of binding to specific peptide featuresbetween arrays. In other words, most features will bind with about thesame relative intensity on one array as they would on another. Thisaspect can be tested with, for example, a Pearson Correlation. However,typically a Pearson Correlation does not provide a complete picturebecause such correlations are strongly weighted by the correspondencebetween the majority of peptide features. The quality of animmunosignature is strongly dictated by the high and low binding peptidefeatures which dictate the shape of the binding distribution (thehistogram of numbers of features that have a certain intensity). Thisdistribution does not consider the peptide-to-peptide correlation, justthe binding properties and dynamic range of the entire data set. Theshape of the distribution can be compared between arrays by using any ofa number of methods, one of which is a Kolmogorov-Smirnov test whichreturns a p-value (a probability value based on the likelihood that theytwo distributions only differed due to random statistical error).

Both the Pearson Correlation and the Kolmogorov-Smirnov test approach1.0 when the assays are similar to one another. By comparing all assaysto all other assays in a batch using these two tests, one can easilydetermine which assays meet both criteria. FIG. 7 shows such an analysisgraphically. The Y-axis is the average Pearson Correlation (the averageof each array against all others is plotted). An array agrees betterwith other arrays the larger this value is. The X-axis is theKolmogorov-Smirnov test (the average of the log of the p-value obtainedfrom this test for each array against all others is plotted). Again, anarray agrees better with other arrays the larger this value is. In thisgraph the actual values shown are simply normalized to fill the screenfor viewing purposes, so only the relative values are meaningful. Alsoshown is a cutoff that corresponds to a particular distance defined bythe two orthogonal parameters (i.e. the distance is the square root ofthe sum of the squares of the two values and is relative to the upperright of the graph where arrays would be in perfect agreement). Thevalue of the cutoff used, depends on the kind of dataset involved.Assays that are either below or to the left of the line are rerun.

What is claimed is:
 1. A method for determining quality and fidelity ofan in situ synthesized immunosignature array, wherein the in situsynthesized immunosignature array comprises an array of peptides, themethod comprising: obtaining a specific binding strength of each peptideof the in situ synthesized immunosignature array, wherein the specificbinding strength is obtained from binding with a first sample, the firstsample comprising a biological sample from a subject; obtaining abackground binding pattern of an immunosignature reference array,wherein the background binding pattern is represented by a mean bindingstrength of empty control regions in the immunosignature referencearray; and measuring a standard deviation of the specific bindingstrength of each peptide compared to the background binding pattern,wherein a measurement of more than three standard deviations establishessynthesis quality and fidelity of the in situ synthesizedimmunosignature array.
 2. The method of claim 1, further comprisingcalculating a correlation value, wherein a correlation value of lessthan a threshold level indicates quality of the immunosignature array.3. The method of claim 2, wherein the correlation value is obtained byKolmogorov-Smirnov test, Pearson Correlation, or Pearson Squared.
 4. Themethod of claim 2, wherein the threshold level is defined as apercentage of a correlation coefficient between assays.
 5. The method ofclaim 1, wherein the mean binding strength of the empty control regionsis subtracted from the specific binding strength of each peptide toestablish a background subtracted value.
 6. The method of claim 1,wherein the specific binding strength of each peptide is divided by themean binding strength of the empty control regions to generate abackground normalized value.
 7. The method of claim 1, wherein theimmunosignature array is a random or partially random peptide array. 8.The method of claim 1, wherein the specific binding strength is obtainedfrom a subset of peptides on the in situ synthesized immunosignaturearray, and the background binding pattern is obtained from the emptycontrol areas in the immunosignature reference array.
 9. The method ofclaim 8, wherein the empty control areas are areas having no peptidespresent, wherein any binding is nonspecific binding to theimmunosignature reference array.
 10. The method of claim 1, wherein thespecific binding strength is obtained from binding with the firstsample, the background binding pattern is obtained from binding with asecond sample, wherein the first sample and the second sample are from asame species.
 11. The method of claim 10, wherein the first sample andthe second sample are not from the same subject.
 12. The method of claim1, wherein the sample is obtained from a human.
 13. The method of claim11, wherein the first sample and the second sample are acquired from ablood sample.
 14. The method of claim 1, wherein the biological sampleis a blood sample, a serum sample, a plasma sample, a urine sample, asputum sample, a mucosal sample, a peritoneal fluid sample, a tissuesample, an exudate or an effusion.